Project Summary/Abstract This project addresses a key issue for both biology and medicine: the discovery of small molecules that can serve as the basis for regulating biological processes and/or developing into therapeutic agents. Bacteria that live in close association with other organisms ? symbiotic bacteria ? produce small molecules to regulate the relations with their hosts and other community members, and researchers are just now beginning to appreciate the pervasiveness of these interactions and the vast array of biologically active small molecules needed to maintain them. This proposal describes three different approaches to access these small molecules and their biological functions. Two focus on specific symbioses, and the third focuses on a general strategy to screen for in vivo virulence factors. 1. The first aim focuses on a previously unrecognized ecological niche, the bacterial symbionts of mushrooms. The motivation for this aim originated in a desire to understand the evolutionary origins of the multilateral symbioses we see today ? the original binary symbioses. Whether this evolutionary scenario is correct or not, the idea of focusing on these bacteria led to some very interesting preliminary results: the tryptorubin system of peptides with an oxidative polycyclization biosynthesis that creates a rigid, strained final product. 2. The second aim focuses on the bacterial symbionts of plants ? the unicellular algae that photosynthesize roughly half of the world's oxygen and fix an equivalent amount of carbon. They also take part in many other global element cycles. These algae require the assistance of their bacterial symbionts to fulfill these functions. The algae and their bacterial symbionts are redistributing due to climate change in ways that we don't fully understand. Some of these algal-bacterial systems involve the production of molecules with impacts or potential impacts on human health. As examples, one makes an amnesic neurotoxin called domoic acid, and another makes a factor that induces a polyploidy phenotype ? a likely cytokinesis inhibitor that could be useful for proliferative diseases. 3. Many of the biologically active molecules that take part in complex symbioses continue to be invisible to our current discovery methods. For example, an active molecule might be made from an unusual metabolite provided by the host of another member of the community, and the `producing' bacteria might be providing a single enzyme. These molecules and processes will be invisible to standard metabolomic or genomic analyses. We have developed an in vivo screen with relatively high throughput that can help identify such processes, and the third specific aim deals with some early proof of concept applications of the screen.